The expression of foreign genes in yeast regulated by the system which causes the expression of ADH II has proved to be quite valuable. Such expression usually involves placing the sequence encoding the heterologous protein under the control of the promoter from the ADH2 gene, or hybrid promoters employing the upstream regulatory region of the ADH2 promoter in combination with a strong yeast promoter, such as the glyceraldehyde-3-phosphate (GAP) promoter. See, e.g, EPO Pub. No. 164,556; EPO Pub. No. 196,056; commonly owned U.S. patent application Ser. No. 868,639, filed May 29, 1986. Expression cassettes for the heterologous protein employing the ADH2 regulatory regions are usually present in the yeast expression host in high copy numbers. While good results are obtained with such expression systems, a continuing need exists to enhance heterologous protein yield.
A number of studies have been published regarding regulation of the ADH2 gene by the yeast ADR1 gene. See, e.g., Shuster et al. (1986) Mol. Cell. Biol. 6:1894-1902; Denis et al. (1983) Mol. Cell. Biol. 3:360-370. Several studies have been published examining the relationship between the expression level of ADR1 and the expression level of native ADH2 genes.
See Irani et al. (1987) Mol. Cell. Biol. 7:1233-1241; Denis (1987) Mol. Gen. Genet. 208:101-106. These studies however, did not demonstrate whether increased ADR1 expression would ultimately improve the yield of a heterologous protein expressed under the control of ADH2 regulatory sequences. For example, it was observed by Irani et al. (1987) that over one hundred copies of ADR1 did not overcome the 3- to 4-fold inhibition in ADH2 transcription caused by multiple ADH2 promoters. It was suggested that this result supports a model of ADH II regulation in which there are positive limiting factors on ADH2 expression other than ADR1. Denis (1987) also reported that increasing ADR1 gene dosage was apparently toxic to the yeast host, resulting in a significant increase in cell doubling time.